**4. The biological research**

The microbiology research of the aflatoxins in China started fairly late. Reviews on biosynthesis of aflatoxins have not been reported until 2003 and researches on aflatoxin resistance were started only since 2001. Molecular biological methods have been carried out in aflatoxin research during the last two years, such as gene chip (microarray) technology used for gene expression studies.

#### **4.1 Biosynthesis of aflatoxins 4.1.1 Process of synthesis**

Based on the improvement in the analysis of the aflatoxins biosynthesis, the aflatoxin biological synthesis process was summarized (Xu & Luo, 2003). In the initial period, with Acetyl-CoA as the original unit and malonyl-CoA as the elongation unit, the reaction is catalysized by polyketide synthase to form the aflatoxin backbone, polyketone. In general, the specific process of the synthesis scheme of AFB1 and AFG1 is from Acetyl-CoA to caproyl-CoA, norsolorinic acid, averantin, averufin (AVF), versiconal hemiacetal acetate, versiconal, versicolorin B, versicolorin A, versicolorin, *O*-methylsterigmatocystin, and finally to AFB1 and

It is widely reported that the aflatoxins cause many human acute intoxication events. For example, farmers in three families ate mildewed rice (the aflatoxin content reached 225.9 μg/kg) in Taiwan province. This event led to 25 persons poisoned and the deaths of three children, among 39 persons involved. There was an explosion of Toxic Hepatitis caused by aflatoxin in 200 villages, 397 persons got the disease and 106 persons dead

After a person ate aflatoxin contaminated food, it may cause fever, abdominal pain, vomiting, more seriously splenohepatomegalia, hepatalgia, skin mucous membrane stained yellow, ascites, edema of lower limbs and dysfunction of liver after 2~3 weeks. The cardiac

The aflatoxin can cause damage to the liver, and the sensitivity of aflatoxin is closely related to animal size, species, gender, age, and nutrition. The aflatoxin can damage to the animal embryo, decrease the liver function, cause a decline in milk and egg production, and decrease animal immunity if infection of micro organism happens repeatedly. During the growth period, animals at young stage are more likely to be infected. The clinical manifestations poisoning include low reproductive capacity, gastrointestinal dysfunction,

According to statistics, the aflatoxins contamination of animal feed in USA led to about 10% financial loss. Besides, the death of livestock results in a severe loss to the agriculture (Zhang, 2008). At the same time, aflatoxins can reduce the production of the food and fiber crops.

The microbiology research of the aflatoxins in China started fairly late. Reviews on biosynthesis of aflatoxins have not been reported until 2003 and researches on aflatoxin resistance were started only since 2001. Molecular biological methods have been carried out in aflatoxin research during the last two years, such as gene chip (microarray) technology

Based on the improvement in the analysis of the aflatoxins biosynthesis, the aflatoxin biological synthesis process was summarized (Xu & Luo, 2003). In the initial period, with Acetyl-CoA as the original unit and malonyl-CoA as the elongation unit, the reaction is catalysized by polyketide synthase to form the aflatoxin backbone, polyketone. In general, the specific process of the synthesis scheme of AFB1 and AFG1 is from Acetyl-CoA to caproyl-CoA, norsolorinic acid, averantin, averufin (AVF), versiconal hemiacetal acetate, versiconal, versicolorin B, versicolorin A, versicolorin, *O*-methylsterigmatocystin, and finally to AFB1 and

dilatation, pulmonary edema, coma, spasm may also occur (Xiao & Xing, 2003).

decline in feed utilization. Moreover, dairy cattle could produce AFM1 and M2.

**3.1.2 Chronic intoxication 3.1.2.1 Correlated event** 

(Wu, 2007).

**3.1.2.2 Symptoms** 

**3.2 Harm to animals** 

**3.3 Financial Ioss** 

**4. The biological research** 

used for gene expression studies.

**4.1 Biosynthesis of aflatoxins 4.1.1 Process of synthesis** 

AFG1; the synthesis of AFB2 and AFG2 is: The front part of the process is the same to AFGB1 and AFG1, the difference is versicolorin B changed to dihydro versicolorin, then to dihydro-Omethylsterigmatocystin, to AFB2 and AFG2.

#### **4.1.2 Factors involved in the synthesis**

The factors closely related to the synthesis of aflatoxins include the genes, enzymes and the environmental conditions. The genes related to the aflatoxin biosythesis were analyzed by the technology of gene chip as well as RT-PCR method (Hu & Xu, 2009). Six abnormally expressed genes were detected. The six genes are *aflA*, *aflE*, *aflF*, *aflR*, *aflT* and *aflX*. According to the result, the different expression level of *aflR* has close correlation with the production of aflatoxin.

Some related factors in the synthesis of aflatoxins were studied (Lu *et al.*, 2010). The results show that several dehydrogenases, peroxidases, cyclases, methyltransferases and oxidoreductase have a key role in the biosynthesis of aflatoxins. The activity of those enzymes affected the yield of aflatoxins directly. On the other hand, the most important environmental factors are carbon and nitrogen source, power of hydrogen, temperature, water activity and plant metabolites.

#### **4.2 The resistant research**

Studies on resistance to aflatoxigenic fungi through molecular biology in China include: the synthesis of artificial antigens, the aflatoxin resistant microorganism and catabolic enzymes, screening of important resistance genes and molecular markers. Aflatoxins are small molecules, thus the immunization of aflatoxins was achieved through coupling with large proteins. With the m-chloroperbenzoic acid (MCPBA) as oxygenant turning the aflatoxin G1 to 8.9- epoxide, a compound AFG1-BSA was obtained after the epoxide coupling with BSA in a two-phase reaction system (Zhang & Li, 2008). Ultraviolet scanning of the compound showed a significant difference comparing with the scanning result of aflatoxin G1 and a different fluorescence intensity between them, which indicated the coupling of BSA and AFG1. This analytical method promoted the study on the preparation of monoclonal antibody and immunoaffinity column.

The mixture of broad bean and wheat flour during fermentation was used to screen antagonistic bacteria against aflatoxigenic *A. flavus* (Gao & Ding, 2010). A strain L4 with strong antifungal activity against the aflatoxin-producing fungus *A. flavus* was selected using agar medium (BAM). According to its morphological, physiological and biochemical characteristics and 16S rRNA gene sequence homology analysis, L4 was identified as *Bacillus subtilis*. When L4 and *A. flavus* were co-cultured for 15 days, the weight of the mycelium and the production of aflatoxin B1 were both significantly lower than those of *A. flavus* cultured without L4. The accumulation of AFB1 was greatly inhibited, the suppression effective ratio was 93.7%. When L4 culture supernatant was mixed with the spore suspension of *A. flavus* at ratio of 1:1 and then inoculated on corn, the germination and growth of *A. flavus* was completely inhibited.

Using the method of filter paper diffusion. a strain of marine microorganism which exhibited highly inhibitory effect on *Aspergillus flavus* was screened (Kong & Liu, 2010). With the aid of 16S rDNA gene sequence, this marine strain was finally identified as a marine strain of *Bacillus megaterium*. Then, its inhibitory effects on mycelium extending, spore germination and aflatoxin biosynthesis of *A. flavus* were further studied. Quantitative analysis kit for aflatoxins (Beacon) was used to determine the concentration of aflatoxin. The

Aflatoxin Contamination and Research in China 29

application of new reagents, the TLC detection becomes a simple and widely used analysis

Using the liquid chromatography (HPLC) method with immuno-affinity column cleanup through post-column derivatization system, aflatoxins can be adsorbed in the immunaffinity column and eluted with organic solvent. The HPLC method with fluorescence detector using post-colummn derivatization system is a commonly used method in different countries (Wang, 2004). This method is more sensitive and accurate. Furthermore, this method is one of

Employing the micro-column method to analyze aflatoxin is first to build up the microcolumn chromatography tube using sample-extracted solvent and then the aflatoxin would be adsorbed by the florisil adsorbent as the alumina absorb the foreign matter. Under 365 nm UV light, the amount of aflatoxin can be calculated by the intensity of the blue-violet light reflected from the compound. This method is accurate, simple, rapid and reproducible.

ELISA is widely used in food and feed industries to determine the content of aflatoxin in food products. Though the ELISA is accurate stable and reproducible, the analysis sometimes shows false positive, or false negative due to enzyme instability and variations of enzymes reaction conditions. So the application of this method in analysis of aflatoxin

Immunochromatography is a kind of immunoassay technique developed in recent years. It is simple, rapid and is suitable for prescreening a large number of samples and for

Efforts to minimize adverse effects of aflatoxins include monitoring, managing and controlling their levels in agricultural products from farm to market and to table. While an association between aflatoxin contamination and inadequate storage conditions has long been recognized, studies have been focused on developing commercial crop cultivars that are resistant to *Aspergillus flavus*, such as peanut varieties Guihua 22 and Yueyou 58. Meantime, selecting the rational planting techniques and harvesting method, reasonable

To date, many countries paid much attention to researches on the development of this method. In China, the new peanut cultivars such as Guihua 22 and Yueyou 58 have been cultivated (Wang, 2004). Since the Vitamin E is an essential factor in the synthesis of

**6.1.1 Selecting the crop cultivar with high level of resistance to aflatoxins** 

the best method for determining aflatoxin in traditional Chinese medicines (Ma, 2007).

However, the micro-column is considered a qualitative method (Xie, 2007).

method. It is still used in China today.

**5.3 Micro-column method** 

**5.2 High Pressure Liquid Chromatography (HPLC)** 

**5.4 Enzyme-linked Immunosorbent assay (ELISA)** 

remains to be improved in future (Ma, 2007).

**6. Prophylactico-therapeutic measures** 

storage conditions and inhibitor are equally important.

**6.1 Control measures in oils and foods** 

**5.5 Immunochromatography** 

analyzing on the spot (Ma, 2007).

results showed that this marine strain exhibited good inhibition to the mycelium growth, spore germination and aflatoxin biosynthesis in *A. flavus*. Eighty-seven percent spore (1×109CFU·mL-1 *B. megaterium*) and 50.75% aflatoxin (1×108CFU·mL-1 *B. magaterium*) were inhibited, compared with control group. The possible mechanism is that some kinds of metabolites secreted by this marine strain can inhibit the mycelium growth and spore germination of *A. flavus*.

Aflatoxin-detoxifizme (ADTZ), being from *Amillariella tabescens*, can effectively decompose aflatoxins. To secretively express ADTZ in *Pichia pastoris* with higher performance, through optimizing the 5'coding region of its cDNA according to the preferred codons of *P. pastoris*  (Zuo & Liu, 2007). Two-step DNA synthesis was used to synthesize the cDNA sequence being optimized of ADTZ (OPT-ADTZ). OPT-ADTZ was inserted in the constitutive plasmid pGAPZαA to construct the recombinant plasmid pNOA. pNOA was linearized and then transformed into *P. pastoris* GS115. Then code-optimized ADTZ was constitutively and secretively expressed in *P. pastoris*. In seed of Balsampear Fruit, the antifungal activity of ribosome inactivating proteins (RIPs) were examined (Liu, 2001). In the research aimed at developing a rapid and reliable screening method for selecting *A. flavus* infection resistance in peanut, two DNA markers closely linked with the resistance to *A. flavus* infection were identified using BSA technique. The two specific fragments were about 440bp and 520bp, respectively. They were named as marker E45M53-440 and E44M5-520 (Lei, 2009). The potential usage of the two markers can be in determining or selecting the resistance to the infection by *A. flavus*.

### **5. Main methods of detection and screening**

Monitoring programs have been established to reduce the risk of aflatoxin consumption by human and animals. Analytical testing methods of large numbers of samples of foodstuffs have been developed for rapid detection of Aflatoxins. Current analytical techniques are more accurate in characterization and quantitation of aflatoxins. These include high pressure liquid chromatography (HPLC), Gas chromatography (GC) and serum assay (ELISA), which are much better than the early thin layer chromatography (TLC) technique (Zhang *et al.*, 2008).

#### **5.1 Thin Layer Chromatography (TLC)**

Thin Layer Chromatography is a chromatography technique used to separate mixtures, which is performed on a sheet of glass, plastic, or aluminum foil coated with a thin layer of adsorbent material usually silica gel, aluminium oxide, or cellulose. It can be used to monitor the progress of molecule migration to identify compounds present in a given substance and to determine the purity of a substance. TLC can also be used on a small semi-preparative scale to separate mixtures of up to a few hundred milligrams. The mixture is not "spotted" on the TLC plate as dots, but rather applied to the plate as a thin even layer horizontally to and just above the solvent level. For small-scale analysis, TLC can be far more efficient in term of time and cost than chromatography. To analyze the amount of aflatoxin in samples by TLC, the smallscale target can be visible at UV light under 365 nm wavelength. According to the intensity, size and color of the spots on TLC plates, the type and its exact form of the compounds can be determined (Xie, 2007). As the TLC analysis is often affected by many factors, the accuracy of this method is poor. With the improvement of extraction and isolation method as well as the

results showed that this marine strain exhibited good inhibition to the mycelium growth, spore germination and aflatoxin biosynthesis in *A. flavus*. Eighty-seven percent spore (1×109CFU·mL-1 *B. megaterium*) and 50.75% aflatoxin (1×108CFU·mL-1 *B. magaterium*) were inhibited, compared with control group. The possible mechanism is that some kinds of metabolites secreted by this marine strain can inhibit the mycelium growth and spore

Aflatoxin-detoxifizme (ADTZ), being from *Amillariella tabescens*, can effectively decompose aflatoxins. To secretively express ADTZ in *Pichia pastoris* with higher performance, through optimizing the 5'coding region of its cDNA according to the preferred codons of *P. pastoris*  (Zuo & Liu, 2007). Two-step DNA synthesis was used to synthesize the cDNA sequence being optimized of ADTZ (OPT-ADTZ). OPT-ADTZ was inserted in the constitutive plasmid pGAPZαA to construct the recombinant plasmid pNOA. pNOA was linearized and then transformed into *P. pastoris* GS115. Then code-optimized ADTZ was constitutively and secretively expressed in *P. pastoris*. In seed of Balsampear Fruit, the antifungal activity of ribosome inactivating proteins (RIPs) were examined (Liu, 2001). In the research aimed at developing a rapid and reliable screening method for selecting *A. flavus* infection resistance in peanut, two DNA markers closely linked with the resistance to *A. flavus* infection were identified using BSA technique. The two specific fragments were about 440bp and 520bp, respectively. They were named as marker E45M53-440 and E44M5-520 (Lei, 2009). The potential usage of the two markers can be in determining or selecting the resistance to the

Monitoring programs have been established to reduce the risk of aflatoxin consumption by human and animals. Analytical testing methods of large numbers of samples of foodstuffs have been developed for rapid detection of Aflatoxins. Current analytical techniques are more accurate in characterization and quantitation of aflatoxins. These include high pressure liquid chromatography (HPLC), Gas chromatography (GC) and serum assay (ELISA), which are much better than the early thin layer chromatography (TLC) technique

Thin Layer Chromatography is a chromatography technique used to separate mixtures, which is performed on a sheet of glass, plastic, or aluminum foil coated with a thin layer of adsorbent material usually silica gel, aluminium oxide, or cellulose. It can be used to monitor the progress of molecule migration to identify compounds present in a given substance and to determine the purity of a substance. TLC can also be used on a small semi-preparative scale to separate mixtures of up to a few hundred milligrams. The mixture is not "spotted" on the TLC plate as dots, but rather applied to the plate as a thin even layer horizontally to and just above the solvent level. For small-scale analysis, TLC can be far more efficient in term of time and cost than chromatography. To analyze the amount of aflatoxin in samples by TLC, the smallscale target can be visible at UV light under 365 nm wavelength. According to the intensity, size and color of the spots on TLC plates, the type and its exact form of the compounds can be determined (Xie, 2007). As the TLC analysis is often affected by many factors, the accuracy of this method is poor. With the improvement of extraction and isolation method as well as the

germination of *A. flavus*.

infection by *A. flavus*.

(Zhang *et al.*, 2008).

**5. Main methods of detection and screening** 

**5.1 Thin Layer Chromatography (TLC)** 

application of new reagents, the TLC detection becomes a simple and widely used analysis method. It is still used in China today.

#### **5.2 High Pressure Liquid Chromatography (HPLC)**

Using the liquid chromatography (HPLC) method with immuno-affinity column cleanup through post-column derivatization system, aflatoxins can be adsorbed in the immunaffinity column and eluted with organic solvent. The HPLC method with fluorescence detector using post-colummn derivatization system is a commonly used method in different countries (Wang, 2004). This method is more sensitive and accurate. Furthermore, this method is one of the best method for determining aflatoxin in traditional Chinese medicines (Ma, 2007).

#### **5.3 Micro-column method**

Employing the micro-column method to analyze aflatoxin is first to build up the microcolumn chromatography tube using sample-extracted solvent and then the aflatoxin would be adsorbed by the florisil adsorbent as the alumina absorb the foreign matter. Under 365 nm UV light, the amount of aflatoxin can be calculated by the intensity of the blue-violet light reflected from the compound. This method is accurate, simple, rapid and reproducible. However, the micro-column is considered a qualitative method (Xie, 2007).

#### **5.4 Enzyme-linked Immunosorbent assay (ELISA)**

ELISA is widely used in food and feed industries to determine the content of aflatoxin in food products. Though the ELISA is accurate stable and reproducible, the analysis sometimes shows false positive, or false negative due to enzyme instability and variations of enzymes reaction conditions. So the application of this method in analysis of aflatoxin remains to be improved in future (Ma, 2007).

#### **5.5 Immunochromatography**

Immunochromatography is a kind of immunoassay technique developed in recent years. It is simple, rapid and is suitable for prescreening a large number of samples and for analyzing on the spot (Ma, 2007).

#### **6. Prophylactico-therapeutic measures**

Efforts to minimize adverse effects of aflatoxins include monitoring, managing and controlling their levels in agricultural products from farm to market and to table. While an association between aflatoxin contamination and inadequate storage conditions has long been recognized, studies have been focused on developing commercial crop cultivars that are resistant to *Aspergillus flavus*, such as peanut varieties Guihua 22 and Yueyou 58. Meantime, selecting the rational planting techniques and harvesting method, reasonable storage conditions and inhibitor are equally important.

#### **6.1 Control measures in oils and foods**

#### **6.1.1 Selecting the crop cultivar with high level of resistance to aflatoxins**

To date, many countries paid much attention to researches on the development of this method. In China, the new peanut cultivars such as Guihua 22 and Yueyou 58 have been cultivated (Wang, 2004). Since the Vitamin E is an essential factor in the synthesis of

Aflatoxin Contamination and Research in China 31

enhance their resistance based on a developed management practice (Tang, 1999b). In recent years, a new technique of utilizing antagonistic microorganisms and the change from wild fermentation to pure fermentation are becoming effective measures to reduce aflatoxin contamination. Similar to crops, the control methods in storage also include the

The practical methods applied for reducing aflatoxin production are: sorting, processing, and the sun light (ultraviolet) sterilization. These traditional meathods have been used since 1995 in China. Significant emphasis has been placed on detoxification of contaminated lots by irradiation, ammonia fumigation, chemical method, oxidants, microbial and enzymatic

Adsorption is the most common method to reduce aflatoxin contamination. For example, the harmful damaging effect of aflatoxins to animals can be reduced by adding several nutrition unactive adsorbents to the feed. Additionally, the adsorbents also can remove a portion of the aflatoxins (Xu *et al.*, 2001). Among which the activated charcoal, mannan oligosarccharide (MOS), aluminosilicate, hydrated aluminosilicate and bentonite have been best studied. However, the positive effect has been observed only in the laboratory. Commercial utilization

With nanomaterial silicate adsorbent added to the feed contaminated by aflatoxins, it can significantly reduce the residual toxin in chicken muscle and liver. This is promising for producing safe animal products that meets international standards (Feng, 2004). The new adsorbent can also effectively reduce its harmful effect on growth, visceral function and the immune system of boilers. In the study on the adsorption of Silicate structure adsorbent NSP in feed of pigs, it was discovered that the absorption function in three forms: adsorption inside layers, adsorption between layers, and adsorption at the edges (Qi, 2002). Absorption of several organic absorbents(KGM, Detoxification substance, Sorbent C) detoxification of AFB1 in animal was studied (Yu, 2007). *In vitro* experiment to study the absorption characteristics through different absorption, different content, different pH, and different temperature indicated that in high temperature, absorption capabilities of the three sorbents are worse than that in low temperature. Absorption capability of KGM is very weak in high temperature, while detoxification substance and sorbent C are obviously better. The three sorbents adsorb better in alkaline than in acidic conditions. But in acidic

Qingdao Agricultural University tested glucomannan to adsorb aflatoxins, the EGM at the concentration of 78.54%, 83.71% and 0.11% showed the best AFB1 adsorption ability, when

Aflatoxin detoxification in peanut and peanut meal by ammonia gas was tested (Liang,2009). Single factor test showed that ammonia temperature, time and water content of samples greatly affected AFB1 degradation. The optimal conditions for best result are 10% amonia by volume, 24% peanuts meal moisture, which gave 100% AFB1 degradation. There

temperature., humidity, and oxygen etc.

pH, sorbent C is worse than the other two.

the concentration of glucomannan is 0.11% (Yu, 2007).

is no detectable AFB1 after ammonia fumigation (Liang,2009).

**7.2 Aflatoxins detoxification by ammonia gas** 

**7.1 Adsorption techniques** 

**7. Methods of degradation and removing** 

methods commonly used in treating corn and peanuts.

of the absorption meathod is rarely used in practical production in China.

aflatoxins, it is important to select a cultivar with low Vitamin E content in seed coat to reduce aflatoxin contamination.

#### **6.1.2 Using reasonable planting techniques and harvesting methods**

It is very important to use suitable planting technique and harvesting method. The unsuitable handling process will cause damage to kernal, which will result in fungal infection and aflatoxin contamination. So during the harvest and storage, any measures that can reduce physical damage to the kernal of crops including insect pest and rats will surely reduce fungal growth and aflatoxin contamination (Wu, 2007).

#### **6.1.3 Using reasonable storage condition**

The storage condition is also an important determinant for reducing aflatoxin contamination. The AFB1 content of dry hot peppers stored under different storage conditions was analyzed to provide the theoretical basis for improving quality of dry hot pepper during storage. The results showed that the storage conditions of low temperature, low moisture content, low relative humidity and sealed package could significantly reduce the occurrence and accumulation of AFB1 in dry hot pepper.

#### **6.1.4 Reasonable adoption of antiseptic**

Utilizing antiseptic agents is effective in preventing aflatoxin contamination. The most commonly used antiseptics are sodium benzoate, sorbitol, propionic acid and propionates. The antiseptic compounds that contain propionic acid, propionates and sorbitol are in high demand. Studies showed that the removing rate of AFG B1 was more than 90% when using ozone to treat AFG B1 in contaminated crops (Luo *et al.*, 2003).

#### **6.2 Control measures in feed**

Except low temperature, low moisture, control of oxygen and using antiseptics, adequate dilution may also prevent the aflatoxin contamination in feed (Wang & Zhang, 2006). However, this method is only applied to the least aflatoxin contaminated situation. The specific dilution methods are to analyze the exact content of aflatoxins and mix those feed for which their toxin content is in the borderline with the unmolded feed materials according to the normal feeding amounts. However, if the diluted feed hasn't been used up soon, it will extend aflatoxin contaminations. The regulating strategy include: based on the fact, adding methionine and electrolyte to improve the hepatic function and increase the natural concentration, especially the level of non-contaminated proteins.

#### **6.3 Control measures in fermented condiment**

Control of aflatoxin in fermented condiment should start from the raw materials. Controlling the key factors such as water activity in the soy bean and the humidity in environment is important. The best parameter should be controlled to keep the water content in soy bean below 13% and the humidity no less than 65% in depositories (Wang & Z., 2009). Combined with the application of antiseptic and ozone treatment the quality of the finished products and semi-finished products can be enhanced.

#### **6.4 Control measure in chinese medicines**

Similar to fermented condiment, the control of aflatoxin in Chinese medicine should also start from the resources. Thus, control measures of the herbal medicine can be taken to

aflatoxins, it is important to select a cultivar with low Vitamin E content in seed coat to

It is very important to use suitable planting technique and harvesting method. The unsuitable handling process will cause damage to kernal, which will result in fungal infection and aflatoxin contamination. So during the harvest and storage, any measures that can reduce physical damage to the kernal of crops including insect pest and rats will surely

The storage condition is also an important determinant for reducing aflatoxin contamination. The AFB1 content of dry hot peppers stored under different storage conditions was analyzed to provide the theoretical basis for improving quality of dry hot pepper during storage. The results showed that the storage conditions of low temperature, low moisture content, low relative humidity and sealed package could significantly reduce

Utilizing antiseptic agents is effective in preventing aflatoxin contamination. The most commonly used antiseptics are sodium benzoate, sorbitol, propionic acid and propionates. The antiseptic compounds that contain propionic acid, propionates and sorbitol are in high demand. Studies showed that the removing rate of AFG B1 was more than 90% when using

Except low temperature, low moisture, control of oxygen and using antiseptics, adequate dilution may also prevent the aflatoxin contamination in feed (Wang & Zhang, 2006). However, this method is only applied to the least aflatoxin contaminated situation. The specific dilution methods are to analyze the exact content of aflatoxins and mix those feed for which their toxin content is in the borderline with the unmolded feed materials according to the normal feeding amounts. However, if the diluted feed hasn't been used up soon, it will extend aflatoxin contaminations. The regulating strategy include: based on the fact, adding methionine and electrolyte to improve the hepatic function and increase the

Control of aflatoxin in fermented condiment should start from the raw materials. Controlling the key factors such as water activity in the soy bean and the humidity in environment is important. The best parameter should be controlled to keep the water content in soy bean below 13% and the humidity no less than 65% in depositories (Wang & Z., 2009). Combined with the application of antiseptic and ozone treatment the quality of the

Similar to fermented condiment, the control of aflatoxin in Chinese medicine should also start from the resources. Thus, control measures of the herbal medicine can be taken to

**6.1.2 Using reasonable planting techniques and harvesting methods** 

reduce fungal growth and aflatoxin contamination (Wu, 2007).

the occurrence and accumulation of AFB1 in dry hot pepper.

ozone to treat AFG B1 in contaminated crops (Luo *et al.*, 2003).

natural concentration, especially the level of non-contaminated proteins.

finished products and semi-finished products can be enhanced.

**6.3 Control measures in fermented condiment** 

**6.4 Control measure in chinese medicines** 

**6.1.3 Using reasonable storage condition** 

**6.1.4 Reasonable adoption of antiseptic** 

**6.2 Control measures in feed** 

reduce aflatoxin contamination.

enhance their resistance based on a developed management practice (Tang, 1999b). In recent years, a new technique of utilizing antagonistic microorganisms and the change from wild fermentation to pure fermentation are becoming effective measures to reduce aflatoxin contamination. Similar to crops, the control methods in storage also include the temperature., humidity, and oxygen etc.

#### **7. Methods of degradation and removing**

The practical methods applied for reducing aflatoxin production are: sorting, processing, and the sun light (ultraviolet) sterilization. These traditional meathods have been used since 1995 in China. Significant emphasis has been placed on detoxification of contaminated lots by irradiation, ammonia fumigation, chemical method, oxidants, microbial and enzymatic methods commonly used in treating corn and peanuts.

#### **7.1 Adsorption techniques**

Adsorption is the most common method to reduce aflatoxin contamination. For example, the harmful damaging effect of aflatoxins to animals can be reduced by adding several nutrition unactive adsorbents to the feed. Additionally, the adsorbents also can remove a portion of the aflatoxins (Xu *et al.*, 2001). Among which the activated charcoal, mannan oligosarccharide (MOS), aluminosilicate, hydrated aluminosilicate and bentonite have been best studied. However, the positive effect has been observed only in the laboratory. Commercial utilization of the absorption meathod is rarely used in practical production in China.

With nanomaterial silicate adsorbent added to the feed contaminated by aflatoxins, it can significantly reduce the residual toxin in chicken muscle and liver. This is promising for producing safe animal products that meets international standards (Feng, 2004). The new adsorbent can also effectively reduce its harmful effect on growth, visceral function and the immune system of boilers. In the study on the adsorption of Silicate structure adsorbent NSP in feed of pigs, it was discovered that the absorption function in three forms: adsorption inside layers, adsorption between layers, and adsorption at the edges (Qi, 2002).

Absorption of several organic absorbents(KGM, Detoxification substance, Sorbent C) detoxification of AFB1 in animal was studied (Yu, 2007). *In vitro* experiment to study the absorption characteristics through different absorption, different content, different pH, and different temperature indicated that in high temperature, absorption capabilities of the three sorbents are worse than that in low temperature. Absorption capability of KGM is very weak in high temperature, while detoxification substance and sorbent C are obviously better. The three sorbents adsorb better in alkaline than in acidic conditions. But in acidic pH, sorbent C is worse than the other two.

Qingdao Agricultural University tested glucomannan to adsorb aflatoxins, the EGM at the concentration of 78.54%, 83.71% and 0.11% showed the best AFB1 adsorption ability, when the concentration of glucomannan is 0.11% (Yu, 2007).

#### **7.2 Aflatoxins detoxification by ammonia gas**

Aflatoxin detoxification in peanut and peanut meal by ammonia gas was tested (Liang,2009). Single factor test showed that ammonia temperature, time and water content of samples greatly affected AFB1 degradation. The optimal conditions for best result are 10% amonia by volume, 24% peanuts meal moisture, which gave 100% AFB1 degradation. There is no detectable AFB1 after ammonia fumigation (Liang,2009).

Aflatoxin Contamination and Research in China 33

human health. "Food Hygiene Control Regulations Article IV" stated clearly that rural and state-owned farms should be organized and guided to harvest in time, threshing, dry, removing impurities, to prevent food mildew pollution during harvesting process. Article VI points out that we should actively carry out the "no worms, no mildew, no rat, and no sparrow" activities. ARTICLE II and III of Prevention Aflatoxin Contamination on Food Hygiene Regulations make clear that we should prevent food mildew and deterioration to achieve the objectives of mould proof and poison removal. Article IV provides that when using grain and oil whose aflatoxin content is higher than allowed level, effective measures must be taken to remove the toxins through technical procedures. The products can only be consumed when the product meets the food safety criterion. Article VI requires that, to ensure infant food safety, a zero tolerance policy should be adopted and food sector should provide non-aflatoxin detectable grain, as materials of infant milk replacer. For aflatoxin monitoring and management, Chinese Health and Quarantine law also established relevant regulations.

Not one or two departments can accomplish aflatoxin control supervision in the process of strengthening food safety supervision system. Team work may play an important and positive role. Management of aflatoxin control mainly involves the following departments.

Due to the problem with aflatoxin contamination during food processing, transportation and marketing process, especially peanuts. The hygiene administrative departments are required to perform some relative control measures on preventing aflatoxin contamination,

Aflatoxin contamination of food is difficult to prevent, therefore, the aspect of food quarantine is particularly important. China has made specific provisions on the highest aflatoxin tolerance amount in all kinds of food. The health and quarantine departments must adopt the advanced science and technology in aflatoxin testing, strictly implement

Because aflatoxin is extremely poisonous substances, it has an aneretic role on human and animal's liver tissue, accompanied with stem cell degeneration and necrosis, eventually result in serious organ damage or even death. Aflatoxins not only damage liver organ in animals, but also affect embryo development in animal. Due to immuno-suppression and recurrent infections aflatoxin contamination in animal feed will reduce milk and eggs production. Experimental results show that aflatoxin toxicities are different depending on animal species, age, and gender. In general, the younger the animals the higher the sensitivity to aflatoxins. Aflatoxins can also pass through food chain to human body through consumption and accumulation in animals. Disease control department should create a healthy environment, maintaining the social stability and national security, improve people's health through the prevention and control of diseases resulted from aflatoxin contamination. Under the leadership of the ministry of health, technological management

**8.2 The organizations involved in aflatoxin control supervision** 

**8.2.1 Hygiene management department** 

**8.2.2 Health and quarantine departments** 

**8.2.3 Disease control department** 

and technical service will be enhanced.

e.g , Food hygiene law of the People's Republic of China.

supervision, to reduce aflatoxin hazard to human health.

#### **7.3 Alkali refining**

Studies by Liuzhou Health and Epidemic Prevention Station and Food Bureau suggested that one part contaminated feed immersed in two parts NaOH should be boiled for 1~2h before feeding to animals. In addition, use lime cream, pure potash and kali to soak aflatoxin contaminated corns for 2~3h, followed by washing in clean water, and drying. The detoxication efficiency can reach 60%~90% (Fan, 2003).

### **7.4 Oxidants**

The treatment with 5% sodium hypochlorite for several seconds could reduce aflatoxin by 98%~100% (Zhang *et al.*, 2004). After analyzing the difference among different time and the products of different places, the ClO2 was effective in detoxicating aflatoxin when the aflatoxin contaminated corns were infused in the 250ug/mL ClO2 for 30~60 min (Zhang & Zhu, 2001). The treatment with 2% sodium bisulfite for 3 days showed best effect on aflatoxin detoxication (Feng, 2002).

#### **7.5 Micro - organisms**

The aflatoxin degradation ability of some food micro-organisms such as the lactic acid bacteria and yeasts was investigated (Zhu & Lin, 2001), (Li *et al.*, 2003). The concentration of aflatoxin, the quantity of fungus and the temperature have a combined effect on the toxin binding ability by lactic acid bacteria. In yeasts, in exponential phase, it showed highest toxin binding ability and the higher concentration of aflatoxin, the higher the binding ability. The enzymatic detoxification of aflatoxin is an effective and safe method, highly selective, no harmful effect on nutrition value and no adverse effect to the treated products (Gong *et al.*, 2004). A new technology on aflatoxin detoxification was developed in recent year. Thoroughly enzymatical hydrolyzation of the peanut meal to achieve full ionization of slightly dissolved aflatoxins from hydrophobic amino acid residues. Then retain the greater part of aflatoxin through successive filtration, thereby make markedly reduction of aflatoxin content (Xu & Luo, 2003).
